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Originally published online as doi:10.2353/jmoldx.2009.080081 on December 18, 2008

Published online before print December 18, 2008
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Journal of Molecular Diagnostics 2009, Vol. 11, No. 1
Copyright © 2009 American Society for Investigative Pathology & Association for Molecular Pathology
DOI: 10.2353/jmoldx.2009.080081

Multi-Probe Real-Time PCR Identification of Common Mycobacterium Species in Blood Culture Broth

Suporn Foongladda*, Suporn Pholwat*, Boonchuay Eampokalap{dagger}, Pattarachai Kiratisin* and Ruengpung Sutthent*

From the Department of Microbiology, * Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok; and the Microbiology Laboratory, {dagger} Bamrasnaradura Institute, Ministry of Public Health, Nonthaburi, Thailand

Mycobacterium tuberculosis complex, M. avium, and M. intracellulare are the most common causes of systemic bacterial infection in AIDS patients. To identify these mycobacterial isolates in primary blood culture broths, we developed a multiple hybridization probe-based real-time PCR assay using the LightCycler system. The primers were designed to amplify a 320-bp fragment of Mycobacterium 16S rRNA genes. Reaction specificity was evaluated using PCR amplification curves along with specific melting temperatures of probes on DNA extracted from 13 Mycobacterium species. In this study, results showed 100% accuracy for the selected bacterial panel. Detection limits were 350, 600, and 650 colony-forming unit (CFU)/ml blood culture broths for M. tuberculosis complex, M. avium, and M. intracellulare, respectively (1 to 2 CFU/reaction). To evaluate clinical applicability, 341 acid-fast bacilli in blood culture broths were analyzed. In total, 327 (96%) were positively identified: 54.5% M. tuberculosis complex, 37.5% M. avium, and 3.8% M. intracellulare. Results can be available within 3 hours of receiving a broth sample, which makes this rapid and simple assay an attractive diagnostic tool for clinical use.






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Copyright © 2009 by the American Society for Investigative Pathology and the Association for Molecular Pathology.